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Anthrax is an important zoonotic disease worldwide that is caused by  Bacillus anthracis , a spore-forming pathogenic bacterium. A rapid and sensitive method to detect  B .  anthracis  is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify  B .  anthracis  by using previously reported molecular-based methods because of the emergence of  B .  cereus , which causes severe extra-intestinal infection, as well as the human pathogenic  B .  thuringiensis , both of which are genetically related to  B .  anthracis . The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a  B .  anthracis  multiplex PCR that can screen for the presence of  B .  anthracis  virulent plasmids and differentiate  B .  anthracis  and its genetically related strains from other  B .  cereus  group species. Six sets of primers targeting a chromosome of  B .  anthracis  and  B .  anthracis -like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of  B .  anthracis  DNA per PCR reaction and was sensitive to  B .  anthracis . The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to  B .  anthracis , which were  B .  cereus  strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated  B .  anthracis  and its genetically related strains from other  B .  cereus  group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting  B .  anthracis .

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species